MOLECULAR CHARACTERIZATIONS OF TWO CYTOCHROME P450 GENES ENCODING CYP6A41 AND CYP6EK1 FROM THE ORIENTAL FRUIT FLY,Bactrocera dorsalis (DIPTERA: TEPHRITIDAE)

Two P450 genes encoding CYP6A41 and CYP6EK1 were cloned from the oriental fruit fly using polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) techniques. CYP6A41 and CYP6EK1 contained open reading frames of 1,530 and 1,524 nucleotides that encode 510 and 508 amino acid residues, respectively. The putative proteins shared 44% identity with each other. Phylogenetic analysis showed that CYP6A41 and CYP6EK1 were most closely related to Ceratitis capitata CYP6A10 and CYP6A subfamily. Expression patterns of the two genes in different geographical populations (Yunnan, Hainan, Dongguang, and Guangzhou), developmental stages (eggs, larvae, pupae, and adults), and tissues (midguts, fat bodies, and Malpighian tubules) were analyzed by real‐time quantitative PCR (RT‐qPCR) methods. The results showed that the expression levels of CYP6EK1 were significantly different among the four populations, but were not different for CYP6A41. Both the expressions of CYP6A41 and CYP6EK1 were development specific and had significantly higher levels in the larval stage. The expression of CYP6A41 did not vary among the midgut, fat body, or Malpighian tubules; however, CYP6EK1 expression was higher in the Malpighian tubules. The results suggest that CYP6A41 and CYP6EK1 might be involved in detoxification of xenobiotic compounds that were harmful to larval flies or development. Moreover, high expression of CYP6EK1 in the Malpighian tubules also implied participation in detoxification.     

Molecular cloning and functional expression of a Drosophila receptor for the neuropeptides capa-1 and -2

The Drosophila Genome Project website contains an annotated gene (CG14575) for a G protein-coupled receptor. We cloned this receptor and found that the cloned cDNA did not correspond to the annotated gene; it partly contained different exons and additional exons located at the 5(')-end of the annotated gene. We expressed the coding part of the cloned cDNA in Chinese hamster ovary cells and found that the receptor was activated by two neuropeptides, capa-1 and -2, encoded by the Drosophila capability gene. Database searches led to the identification of a similar receptor in the genome from the malaria mosquito Anopheles gambiae (58% amino acid residue identities; 76% conserved residues; and 5 introns at identical positions within the two insect genes). Because capa-1 and -2 and related insect neuropeptides stimulate fluid secretion in insect Malpighian (renal) tubules, the identification of this first insect capa receptor will advance our knowledge on insect renal function.     

cDNA cloning, expression, and characterization of an arylphorin-like hexameric storage protein, AgeHex2, from the mulberry longicorn beetle, Apriona germari

An arylphorin-like hexameric storage protein, AgeHex2, cDNA was cloned from the mulberry longicorn beetle, Apriona germari (Coleoptera, Cerambycidae), larval cDNA library. The complete cDNA sequence of AgeHex2 is comprised of 2,088 bp encoding 696 amino acid residues. The AgeHex2 had four potential N-glycosylation sites. The AgeHex2 contained the highly conserved two larval storage protein signature motifs. The deduced protein sequence of AgeHex2 showed high homology with A. germari hexamerin1 (51% amino acid identity), Tenebrio molitor hexamerin2 (49% amino acid identity), T. molitor early-staged encapsulation inducing protein (43% amino acid identity), and Leptinotarsa decemlineata diapause protein1 (43% amino acid identity). Phylogenetic analysis further confirmed the AgeHex2 is more closely related to coleopteran hexamerins than to the other insect storage proteins. Northern blot analysis confirmed that the AgeHex2 showed fat body-specific expression. The cDNA encoding AgeHex2 was expressed as a 75-kDa protein in the baculovirus-infected insect cells. Furthermore, N-glycosylation of the recombinant AgeHex2 was revealed by tunicamycin to the recombinant virus-infected Sf9 cells, demonstrating that the AgeHex2 is N-glycosylated. Western blot analysis using the polyclonal antiserum against recombinant AgeHex2 indicated that the AgeHex2 corresponds to a 75-kDa storage protein present in the A. germari larval hemolymph.     

Identification,functional characterization and expression of a LAT type amino acid transporter from the mosquito Aedes aegypti

We isolated two cDNAs from the mosquito Aedes aegypti, an L-amino acid transporter (AeaLAT) and a CD98 heavy chain (AeaCD98hc). Expression of AeaCD98hc or AeaLAT alone in Xenopus oocyte did not induce amino acid transport activity. However, co-expression of AeaCD98hc and AeaLAT, which are postulated to form a heterodimer protein linked through a disulfide bond, showed significant increase in amino acid transport activity. This heterodimeric protein showed uptake specificity for large neutral and basic amino acids. Small acidic neutral amino acids were poor substrates for this transporter. Neutral amino acid (leucine) uptake activity was partially Na+ dependent, because leucine uptake was approximately 44% lower in the absence of Na+ than in its presence. However, basic amino acid (lysine) uptake activity was completely Na+ independent at pH of 7.4. Extracellular amino acid concentration could be the main factor that determined amino acid transport. These results suggest the heteromeric protein is likely a uniporter mediating diffusion of amino acids in the absence of ions. The AeaLAT showed high level expression in the gastric caeca, Malpighian tubules and hindgut of larvae. In caeca and hindgut expression was in the apical cell membrane. However, in Malpighian tubules and in midgut, the latter showing low level expression, the transporter was detected in the basolateral membrane. This expression profile supports the conclusion that this AeaLAT is a nutrient amino acid transporter.     

Cloning and sequencing of cDNAs coding for the human intra-acrosomal antigen SP-10.

cDNAs coding for the intra-acrosomal protein SP-10 were cloned and characterized as a first step in understanding the expression of this antigen during spermatogenesis. Three overlapping SP-10-specific cDNAs were isolated from a human testes cDNA expression library. These cDNAs hybridized to a 1.35-kb mRNA that was present in human testes but was not found in liver or placenta. Complete sequencing of these cDNAs, designated SP-10-5, SP-10-8, and SP-10-10, produced an 1117-bp sequence containing a 265-amino acid-coding region for the SP-10 protein. Hydrophobicity plots generated from the deduced amino acid sequence showed a very hydrophobic amino terminus characteristic of a signal peptide. Sequence data showed that three different amino acid repeats occurred a total of 16 times in the central third of the SP-10 protein. Interestingly, cDNA SP-10-10 has an internal 57-base pair (19 amino acids) in-frame deletion that is not present in SP-10-5, suggesting that alternative splicing generates more than one SP-10 mRNA. The SP-10 protein appears to be a unique acrosomal protein, based on previous immunohistological data and the observation that SP-10 cDNA sequences did not show any significant homology to other sequences found in the Genbank, National Biomedical Research Foundation, or Swiss sequence banks. A recombinant SP-10 fusion protein was produced in an Escherichia coli expression vector and used to generate a polyclonal antiserum. This antiserum stained the acrosomal cap in situ and reacted with a similar set of peptides on Western blots as did a monoclonal antibody to SP-10.     

Partial purification of a novel insect antidiuretic factor from the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), which acts on Malpighian tubules

In this study, we report for the first time the presence of an antidiuretic factor in the head of the Colorado potato beetle (Leptinotarsa decemlineata (Say)) which acts directly on Malpighian tubules. Biologically active fractions were isolated from the head and separated using molecular weight filtration and high-performance liquid chromatography (HPLC). The resulting fractions were tested for their antidiuretic activity on single isolated Malpighian tubules. Antidiuretic activity was found in the 25% acetonitrile Sep-Pak fraction and the Cn-2 (3000-10,000 MW) and Cn-3 (<3000 MW) fractions, suggesting that the antidiuretic factor was probably a peptide of 25 to 50 amino acids. The antidiuretic factor was very potent, since after five successive fractionations on two different HPLC columns, a high level of inhibition (63%) of fluid secretion by Malpighian tubules could be observed at low dose (0.14 head-equivalent/microl). The antidiuretic factor isolated from the head of the Colorado potato beetle was not affected by repeated freezing and thawing but was sensitive to heat. The differences observed between the Colorado potato beetle antidiuretic factor and other insect diuretic and antidiuretic factors may indicate the possibility of a novel family of water regulation hormones in insects.     

Expression patterns of genes encoding proteins with peritrophin A domains and protein localization in Mamestra configurata

Genes encoding three proteins (McPPAD1-3) with peritrophin A chitin-binding domains (PADs) were identified from a Mamestra configurata larval midgut cDNA library. In addition to midgut, McPPAD1-3 and a previously identified gene encoding the peritrophin, McPM1, were expressed in foregut, hindgut, Malpighian tubules, tracheae, fat body and cuticle; however, the corresponding McPPAD proteins exhibited different localization patterns. McPPAD1 was restricted to the digestive tract and Malpighian tubules, McPPAD2 to Malpighian tubules, and McPPAD3 to the foregut, midgut, hindgut, tracheae and cuticle. Protein fold recognition analysis using tachycitin as a guide structure modelled the McPPAD1 PADs, but not McPPAD2 or McPPAD3 PADs. The McPPAD1 PADs were predicted to contain three anti-parallel β-sheets and a hevein-like fold that form a chitin-binding pocket containing two hydrophobic R-groups in a sandwich-like orientation.     

Uric acid levels during last larval instar of Manduca sexta, an abrupt transition from excretion to storage in fat body

Levels of uric acid in the whole body of the tobacco hornworm, Manduca sexta increased steadily for the 9 days of the fifth instar. However, concentrations in the haemolymph were lowest during the transition from the feeding stage to the wandering stage (days 3, 4), the time when there was a switch from uric acid excretion by the Malpighian tubule-hindgut system to storage in the fat body. Haemolymph volumes, determined for larvae between 2 and 6 days into the fifth instar by isotope dilution with [¹⁴C]-inulin, were used to calculate rates of incorporation of uric acid into Malpighian tubules and fat body of larvae injected with [¹⁴C]-uric acid. These labelling studies indicated that the Malpighian tubules ceased to remove uric acid from the haemolymph some time between the last 6 hr of day 3 of the fifth instar and the first 18 hr of day 4. At the same period, fat body removed significant quantities of uric acid from the haemolymph. The times of initial decreases and increases in levels of uric acid in haemolymph and fat body, respectively, indicated that storage in the fat body started before cessation of elimination via the Malpighian tubule-hindgut system.     

Excretory role of the midgut in larvae of the tobacco hornworm, Manduca sexta (L.).

Caterpillars of Manduca sexta use two distinct transport mechanisms for the excretion of dyes. One pump (Type A) has a high affinity for acid (anionic) dyes and occurs in the midgut and medial Malpighian tubules. Acid dyes accumulate rapidly in the lumen of the midgut while the Malpighian tubules appear to play only a minor role in the excretion of these dyes. The other pump (Type B) excretes basic (cationic) dyes and is located primarily in the proximal Malpighian tubules. Evidence is presented that hippuric acid competes with acid dyes for excretion by both midgut and Malpighian tubules. After the final-instar larva purges its gut the ability of the midgut and Malpighian tubules to excrete dyes gradually decreases. Sixty hours after the purge only the Malpighian tubules retain some dye excreting activity.     

The control of diuresis in the tsetse fly Glossina austeni: a preliminary investigation of the diuretic hormone.

The rate of secretion of the Malpighian tubules of Glossina austeni is controlled by a diuretic hormone. This hormone is present in the nervous tissue of the fly together with a degradative enzyme that can be activated by boiling. It is demonstrated that the Malpighian tubules are able to destroy the diuretic hormone; they may therefore participate in the control of diuresis. The diuretic hormone appears to be a heat-stable, non-dialysable, alcohol-soluble molecule, containing amino acid, glucose and sialic acid residues.